RESUMEN
We examined the permeabilization of lipid bilayers by the beta-sheet, cyclic antimicrobial decapeptide gramicidin S (GS) in phospholipid bilayers formed either by mixtures of zwitterionic diphytanoylphosphatidylcholine and anionic diphytanoylphosphatidylglycerol or by single zwitterionic unsaturated phosphatidylcholines having various hydrocarbon chain lengths, with and without cholesterol. In the zwitterionic bilayers formed by the phosphatidylcholines, without or with cholesterol, the peptide concentrations and membrane potentials required to initiate membrane permeabilization vary little as function of bilayer thickness and cholesterol content. In all the systems tested, the GS-induced transient ion conductance events exhibit a broad range of conductances, which are little affected by the bilayer composition or thickness. In the zwitterionic phosphatidylcholine bilayers, the effect of GS does not depend on the polarity of the transmembrane potential; however, in bilayers formed from mixtures of phosphatidylcholines and anionic phospholipids, the polarity of the transmembrane potential becomes important, with the GS-induced conductance events being much more frequent when the GS-containing solution is positive relative to the GS-free solution. Overall, these results suggest that GS does not form discrete, well-defined, channel-like structures in phospholipid bilayers, but rather induces a wide variety of transient, differently sized defects which serve to compromise the bilayer barrier properties for small electrolytes.
Asunto(s)
Antiinfecciosos/farmacología , Gramicidina/farmacología , Canales Iónicos/química , Membrana Dobles de Lípidos/química , Fosfolípidos/química , Conformación Molecular , Estructura Molecular , PermeabilidadRESUMEN
Tryptophan residues often are found at the lipid-aqueous interface region of membrane-spanning proteins, including ion channels, where they are thought to be important determinants of protein structure and function. To better understand how Trp residues modulate the function of membrane-spanning channels, we have examined the effects of Trp replacements on the structure and function of gramicidin A channels. Analogues of gramicidin A in which the Trp residues at positions 9, 11, 13, and 15 were sequentially replaced with Gly were synthesized, and the three-dimensional structure of each analogue was determined using a combination of two-dimensional NMR techniques and distance geometry-simulated annealing structure calculations. Though Trp --> Gly substitutions destabilize the beta6.3-helical gA channel structure, it is possible to determine the structure of analogues with Trp --> Gly substitutions at positions 11, 13, and 15, but not for the analogue with the Trp --> Gly substitution at position 9. The Gly11-, Gly13-, and Gly15-gA analogues form channels that adopt a backbone fold identical to that of native gramicidin A, with only small changes in the side chain conformations of the unsubstituted residues. Single-channel current measurements show that the channel function and lifetime of the analogues are significantly affected by the Trp --> Gly replacements. The conductance variations appear to be caused by sequential removal of the Trp dipoles, which alter the ion-dipole interactions that modulate ion movement. The lifetime variations did not appear to follow a clear pattern.
Asunto(s)
Glicina/química , Gramicidina/metabolismo , Sustitución de Aminoácidos , Gramicidina/química , Modelos Moleculares , Resonancia Magnética Nuclear BiomolecularRESUMEN
At submicromolar concentrations, capsaicin specifically activates the TRPV1 receptor involved in nociception. At micro- to millimolar concentrations, commonly used in clinical and in vitro studies, capsaicin also modulates the function of a large number of seemingly unrelated membrane proteins, many of which are similarly modulated by the capsaicin antagonist capsazepine. The mechanism(s) underlying this widespread regulation of protein function are not understood. We investigated whether capsaicin could regulate membrane protein function by changing the elasticity of the host lipid bilayer. This was done by studying capsaicin's effects on lipid bilayer stiffness, measured using gramicidin A (gA) channels as molecular force-transducers, and on voltage-dependent sodium channels (VDSC) known to be regulated by bilayer elasticity. Capsaicin and capsazepine (10-100 microM) increase gA channel appearance rate and lifetime without measurably altering bilayer thickness or channel conductance, meaning that the changes in bilayer elasticity are sufficient to alter the conformation of an embedded protein. Capsaicin and capsazepine promote VDSC inactivation, similar to other amphiphiles that decrease bilayer stiffness, producing use-dependent current inhibition. For capsaicin, the quantitative relation between the decrease in bilayer stiffness and the hyperpolarizing shift in inactivation conforms to that previously found for other amphiphiles. Capsaicin's effects on gA channels and VDSC are similar to those of Triton X-100, although these amphiphiles promote opposite lipid monolayer curvature. We conclude that capsaicin can regulate VDSC function by altering bilayer elasticity. This mechanism may underlie the promiscuous regulation of membrane protein function by capsaicin and capsazepine-and by amphiphilic drugs generally.
Asunto(s)
Capsaicina/farmacología , Membrana Dobles de Lípidos , Canales de Sodio/efectos de los fármacos , Capsaicina/análogos & derivados , Gramicidina/farmacología , Cinética , Técnicas de Placa-Clamp , Dispersión de RadiaciónRESUMEN
Proteins, including ion channels, often are described in terms of some average structure and pictured as rigid entities immersed in a featureless solvent continuum. This simplified view, which provides for a convenient representation of the protein's overall structure, incurs the risk of deemphasizing important features underlying protein function, such as thermal fluctuations in the atom positions and the discreteness of the solvent molecules. These factors become particularly important in the case of ion movement through narrow pores, where the magnitude of the thermal fluctuations may be comparable to the ion pore atom separations, such that the strength of the ion channel interactions may vary dramatically as a function of the instantaneous configuration of the ion and the surrounding protein and pore water. Descriptions of ion permeation through narrow pores, which employ static protein structures and a macroscopic continuum dielectric solvent, thus face fundamental difficulties. We illustrate this using simple model calculations based on the gramicidin A and KcsA potassium channels, which show that thermal atomic fluctuations lead to energy profiles that vary by tens of kcal/mol. Consequently, within the framework of a rigid pore model, ion-channel energetics is extremely sensitive to the choice of experimental structure and how the space-dependent dielectric constant is assigned. Given these observations, the significance of any description based on a rigid structure appears limited. Creating a conducting channel model from one single structure requires substantial and arbitrary engineering of the model parameters, making it difficult for such approaches to contribute to our understanding of ion permeation at a microscopic level.
Asunto(s)
Proteínas Bacterianas/química , Gramicidina/química , Activación del Canal Iónico , Modelos Químicos , Modelos Moleculares , Canales de Potasio/química , Solventes/química , Simulación por Computador , Transporte Iónico , Permeabilidad , Porosidad , Conformación Proteica , Estructura Secundaria de Proteína , Relación Estructura-ActividadRESUMEN
In tissue or nerve injury, proinflammatory mediators are released that can modulate a variety of ion channels found in nociceptors. The changes in channel activity, which primarily occurs through changes in intracellular pathways, may lead to the pathological states of hyperalgesia and allodynia. To understand further the regulatory mechanisms underlying the changes in channel activity, we used whole cell patch-clamp recordings from capsaicin-sensitive nociceptive neurons in rat trigeminal ganglion neurons to examine how the cGMP-dependent pathways may regulate ion channel function. Addition of the 8-(4-chlorophenylthio)-3',5' (CPT)-cGMP, a membrane permeant modulator of ion channels, decreased the number of evoked action potentials by 36% and inhibited the tetrodotoxin-resistant (TTX-R) sodium currents and IA potassium currents by 37 and 32%, respectively. Delayed rectifier potassium (IK) currents were unaffected, suggesting that the effects of CPT-cGMP are unlikely to arise from a nonspecific effect on channel activity as a consequence of the adsorption of amphipathic CPT-cGMP molecules to the membrane's bilayer component. This conclusion was reinforced by the lack of changes in gramicidin A channel function in the presence of CTP-cGMP. In summary, the activation of the cGMP-dependent pathways reduces nociceptor excitability, in part, by decreasing the activity of voltage-gated TTX-R sodium channels. This pathway may be a target for efforts to produce selective analgesics.
Asunto(s)
GMP Cíclico/análogos & derivados , GMP Cíclico/farmacología , Activación del Canal Iónico/fisiología , Canales Iónicos/fisiología , Nociceptores/fisiología , 8-Bromo Monofosfato de Adenosina Cíclica/farmacología , Potenciales de Acción/efectos de los fármacos , Animales , Axones/fisiología , Capsaicina/farmacología , Células Cultivadas , Electrofisiología , Gramicidina/farmacología , Activación del Canal Iónico/efectos de los fármacos , Canales Iónicos/efectos de los fármacos , Membrana Dobles de Lípidos , Conducción Nerviosa/efectos de los fármacos , Neuronas/efectos de los fármacos , Nociceptores/efectos de los fármacos , Umbral del Dolor/efectos de los fármacos , Umbral del Dolor/fisiología , Técnicas de Placa-Clamp , Bloqueadores de los Canales de Potasio/farmacología , Ratas , Ratas Sprague-Dawley , Bloqueadores de los Canales de Sodio/farmacología , Tetrodotoxina/farmacología , Ganglio del Trigémino/citología , Ganglio del Trigémino/efectos de los fármacos , Ganglio del Trigémino/fisiologíaRESUMEN
The mechanism(s) underlying the sorting of integral membrane proteins between the Golgi complex and the plasma membrane remain uncertain because no specific Golgi retention signal has been found. Moreover one can alter a protein's eventual localization simply by altering the length of its transmembrane domain (TMD). M. S. Bretscher and S. Munro (SCIENCE: 261:1280-1281, 1993) therefore proposed a physical sorting mechanism based on the hydrophobic match between the proteins' TMD and the bilayer thickness, in which cholesterol would regulate protein sorting by increasing the lipid bilayer thickness. In this model, Golgi proteins with short TMDs would be excluded from cholesterol-enriched domains (lipid rafts) that are incorporated into transport vesicles destined for the plasma membrane. Although attractive, this model remains unproven. We therefore evaluated the energetic feasibility of a cholesterol-dependent sorting process using the theory of elastic liquid crystal deformations. We show that the distribution of proteins between cholesterol-enriched and cholesterol-poor bilayer domains can be regulated by cholesterol-induced changes in the bilayer physical properties. Changes in bilayer thickness per se, however, have only a modest effect on sorting; the major effect arises because cholesterol changes also the bilayer material properties, which augments the energetic penalty for incorporating short TMDs into cholesterol-enriched domains. We conclude that cholesterol-induced changes in the bilayer physical properties allow for effective and accurate sorting which will be important generally for protein partitioning between different membrane domains.
Asunto(s)
Membrana Celular/química , Colesterol/química , Aparato de Golgi/química , Membrana Dobles de Lípidos/química , Proteínas de la Membrana/química , Modelos Biológicos , Modelos Moleculares , Colesterol/farmacología , Simulación por Computador , Transferencia de Energía , Estudios de Factibilidad , Interacciones Hidrofóbicas e Hidrofílicas , Sustancias Macromoleculares , Movimiento (Física) , Conformación Proteica , Transporte de Proteínas/efectos de los fármacos , Transporte de Proteínas/fisiologíaRESUMEN
To further investigate the effect of single amino acid substitution on the structure and function of the gramicidin channel, an analogue of gramicidin A (GA) has been synthesized in which Trp(15) is replaced by Gly in the critical aqueous interface and cation binding region. The structure of Gly(15)-GA incorporated into SDS micelles has been determined using a combination of 2D-NMR spectroscopy and molecular modeling. Like the parent GA, Gly(15)-GA forms a dimeric channel composed of two single-stranded, right-handed beta(6.3)-helices joined by hydrogen bonds between their N-termini. The replacement of Trp(15) by Gly does not have a significant effect on backbone structure or side chain conformations with the exception of Trp(11) in which the indole ring is rotated away from the channel axis. Measurement of the equilibrium binding constants and Delta G for the binding of monovalent cations to GA and Gly(15)-GA channels incorporated into PC vesicles using (205)Tl NMR spectroscopy shows that monovalent cations bind much more weakly to the Gly(15)-GA channel entrance than to GA channels. Utilizing the magnetization inversion transfer NMR technique, the transport of Na(+) ions through GA and Gly(15)-GA channels incorporated into PC/PG vesicles has been investigated. The Gly(15) substitution produces an increase in the activation enthalpy of transport and thus a significant decrease in the transport rate of the Na(+) ion is observed. The single-channel appearances show that the conducting channels have a single, well-defined structure. Consistent with the NMR results, the single-channel conductances are reduced by 30% and the lifetimes by 70%. It is concluded that the decrease in cation binding, transport, and conductance in Gly(15)-GA results from the removal of the Trp(15) dipole and, to a lesser extent, the change in orientation of Trp(11).
Asunto(s)
Glicina/química , Gramicidina/química , Membrana Dobles de Lípidos/química , Micelas , Fosfatidilcolinas/química , Fosfatidilgliceroles/química , Secuencia de Aminoácidos , Sitios de Unión , Transporte Biológico/fisiología , Cationes Monovalentes/química , Glicina/fisiología , Datos de Secuencia Molecular , Resonancia Magnética Nuclear Biomolecular , Técnicas de Placa-Clamp , Dodecil Sulfato de Sodio/química , Relación Estructura-Actividad , Talio , TermodinámicaRESUMEN
To examine the structural and functional importance of backbone amide groups in ion channels for subunit folding, hydrogen bonding, ion solvation, and ion permeation, we replaced the peptide bond between Val(1) and Gly(2) in gramicidin A by an ester bond. The substitution is at the junction between the two channel subunits, where it removes an intramolecular hydrogen bond between the NH of Gly(2) and the C==O of Val(7) and perturbs an intermolecular hydrogen bond between the C==O of Val(1) in one subunit and the NH of Ala(5) in the other subunit. The substitution thus perturbs not only subunit folding but also dimer assembly, in addition to any effects on ion permeation. This backbone modification has large effects on channel function: It alters channel stability, as monitored by the channel forming ability and channel lifetime, and ion permeability, as monitored by changes in single-channel conductance and cation permeability ratios. In fact, the homodimeric channels, with two ester-containing subunits, have lifetimes so short that it becomes impossible to characterize them in any detail. The peptide --> ester substitution, however, does not affect the basic subunit fold because heterodimeric channels can form between a subunit with an ester bond and a native subunit. These heterodimeric channels, with only a single ester bond, are more easily characterized; the lone ester reduces the single-channel conductance about 4-fold and the lifetime about 200-fold as compared to the native homodimeric channels. The altered channel function results from a perturbation/disruption of the hydrogen bond network that stabilizes the backbone, as well as the membrane-spanning dimer, and that forms the lining of the ion-conducting pore. Molecular dynamics simulations show the expected destabilization of the modified heterodimeric or homodimeric channels, but the changes in backbone structure and dynamics are remarkably small. The ester bond is somewhat unstable, which precluded further structural characterization. The lability also led to a hydrolysis product that terminates with an alcohol and lacks formyl-Val. Symmetric channels formed by the hydrolyzed product again have short lifetimes, but the channels are distinctly different from those formed by the ester gramicidin A. Furthermore, well-behaved asymmetric channels form between the hydrolysis product and reference subunits that have either an L- or a D-residue at the formyl-NH-terminus.
Asunto(s)
Sustitución de Aminoácidos , Antibacterianos/química , Gramicidina/química , Canales Iónicos/química , Canales Iónicos/fisiología , Amidas/química , Secuencia de Aminoácidos , Antibacterianos/síntesis química , Cationes , Dimerización , Dipéptidos/síntesis química , Dipéptidos/química , Conductividad Eléctrica , Ésteres , Gramicidina/síntesis química , Enlace de Hidrógeno , Hidrólisis , Potenciales de la Membrana , Datos de Secuencia Molecular , Conformación Proteica , Estructura Secundaria de Proteína , Relación Estructura-ActividadRESUMEN
The energetics of protein-induced bilayer deformation in systems with finite monolayer equilibrium curvature were investigated using an elastic membrane model. In this model the bilayer deformation energy delta G(def) has two major components: a compression-expansion component and a splay-distortion component, which includes the consequences of a bilayer curvature frustration due to a monolayer equilibrium curvature, c(0), that is different from zero. For any choice of bilayer material constants, the value of delta G(def) depends on global bilayer properties, as described by the bilayer material constants, as well as the energetics of local lipid packing adjacent to the protein. We introduce this dependence on lipid packing through the contact slope, s, at the protein-bilayer boundary. When c(0) = 0, delta G(def) can be approximated as a biquadratic function of s and the monolayer deformation at the protein/bilayer boundary, u(0): delta G(def) = a(1)u(0)(2) + a(2)u(0)s + a(3)s(2), where a(1), a(2), and a(3) are functions of the bilayer thickness, the bilayer compression-expansion and splay-distortion moduli, and the inclusion radius (this expression becomes exact when the Gaussian curvature component of delta G(def) is negligible). When c(0) not equal 0, the curvature frustration contribution is determined by the choice of boundary conditions at the protein-lipid boundary (by the value of s), and delta G(def) is the sum of the energy for c(0) = 0 plus the curvature frustration-dependent contribution. When the energetic penalty for the local lipid packing can be ignored, delta G(def) will be determined only by the global bilayer properties, and a c(0) > 0 will tend to promote a local inclusion-induced bilayer thinning. When the energetic penalty for local lipid packing is large, s will be constrained by the value of c(0). In a limiting case, where s is determined only by geometric constraints imposed by c(0), a c(0) > 0 will impede such local bilayer thinning. One cannot predict curvature effects without addressing the proper choice of boundary conditions at the protein-bilayer contact surface.
Asunto(s)
Membrana Dobles de Lípidos/química , Fenómenos Biofísicos , Biofisica , Técnicas In Vitro , Proteínas de la Membrana/química , Modelos Químicos , Conformación Molecular , TermodinámicaRESUMEN
The interactions between an aliphatic or phenyl side chain and an indole ring in a phospholipid environment were investigated by synthesizing and characterizing gramicidins in which Trp(9) was ring-labeled and D-Leu(10) was replaced by D-Val, D-Ala, or D-Phe. All three analogues form conducting channels, with conductances that are lower than that of gramicidin A (gA) channels. The channel lifetimes vary by less than 50% from that of gA channels. Circular dichroism spectra and size-exclusion chromatography show that the conformation of each analogue in dimyristoylphosphatidylcholine (DMPC) vesicles is similar to the right-handed beta(6.3)-helical conformation that is observed for gA. (2)H NMR spectra of oriented samples in DMPC show large changes for the Trp(9) ring when residue 10 is modified, suggesting a steric interaction between D-Leu(10) and Trp(9), in agreement with previous acylation studies (R. E. Koeppe II et al. (1995) Biochemistry 34, 9299-9307). The outer quadrupolar splitting for Trp(9) is unchanged with D-Phe(10), at approximately 153 kHz, but increases by approximately 25 kHz with D-Val(10) and decreases by approximately 10 kHz with D-Ala(10). With D-Ala(10) or D-Val(10), the outer resonance splits into two in a temperature-dependent manner. The NMR spectra indicate that the side chain torsion angles chi1 and chi2 for Trp(9) change when residue 10 is substituted. The changes in chi1 are small, in all cases less than 10 degrees, as is Deltachi2 when D-Ala(10) is introduced, but with D-Val(10) and D-Phe(10) Deltachi2 is at least 25 degrees. We conclude that D-Leu(10) helps to stabilize an optimal orientation of Trp(9) in gA channels in lipid bilayers and that changes in Trp orientation alter channel conductance and lifetime without affecting the basic channel fold.
Asunto(s)
Gramicidina/química , Canales Iónicos/química , Leucina/química , Triptófano/química , Secuencia de Aminoácidos , Sustitución de Aminoácidos , Cromatografía en Gel , Dicroismo Circular , Deuterio , Gramicidina/análogos & derivados , Membrana Dobles de Lípidos/química , Datos de Secuencia Molecular , Resonancia Magnética Nuclear Biomolecular , Fosfatidilcolinas/química , Conformación ProteicaRESUMEN
A reference method is described for the determination of the substance concentration of ionized calcium in plasma by which ionized calcium (free or unbound) may be reliably determined on the basis of calibration with aqueous solutions with known concentration of ionized calcium. The composition of the calibration solutions is chosen such that the activity coefficient of the calcium ion is assumed to be identical both in the calibration solutions and in "normal" plasma, i.e. by convention, the ionic strength (Im) is 0.160 mol/kg. The convention is adopted of reporting ionized calcium measurements as concentration expressed as mmol/l. The proposed reference method for ionized calcium measurement in plasma is based on the use of a cell consisting of an external reference electrode with a saturated potassium chloride liquid/liquid junction in combination with a calcium ion-selective membrane electrode of defined construction and performance. Procedures for using the reference cell and a protocol for sample measurement are described. The preparation of the calibration solutions to be used are described in detail in Appendix A, secondary calibration solutions and check standards in Appendix B, and reference cell vessel design in Appendix C.
Asunto(s)
Sangre , Calcio/sangre , Química Clínica/instrumentación , Química Clínica/métodos , Iones/sangre , Plasma , Estándares de Referencia , Cloruro de Calcio/farmacología , Calibración , Cationes , Electrodos , Humanos , Reproducibilidad de los Resultados , Temperatura , Factores de TiempoRESUMEN
Mutations and chemical substitutions of amino acid side chains and backbone atoms have proved vital for understanding the folding, structure and function of gramicidin channels in phospholipid membranes. The channel's pore is lined by peptide backbone groups; their importance for channel structure and function is shown by a single amide-to-ester replacement within the backbone, which greatly reduces the resulting channel conductance and lifetime. The four tryptophans and the intervening leucines together govern the formation and dissociation of conducting channels from single-stranded subunits. Conducting double-stranded gramicidin conformations (channels) occur rarely in membranes--except when the sequence has been altered to permit special arrangements of tryptophans or (infrequently) in unusually thick membranes. The tryptophans anchor the single-stranded channels to the membrane/solution interface, and the indole dipoles promote cation transport through the channels. Removal of any indole dipole reduces ion conductance; whereas 5-fluorination of an indole, which increases its dipole moment, enhances ion conductance. Some sequence changes at the formyl-NH-terminus (in the membrane interior, away from the tryptophans), including fluorination of the formyl-NH-terminal valine, introduce voltage-dependent channel gating. Gramicidin channels are not just static conductors, but also dynamic entities whose structure and function can be manipulated by backbone and side chain modifications.
Asunto(s)
Antibacterianos/química , Gramicidina/química , Canales Iónicos , Secuencia de Aminoácidos , Diseño de Fármacos , Modelos Moleculares , Datos de Secuencia Molecular , Mutación , Estructura Secundaria de ProteínaRESUMEN
Hydrophobic interactions between a bilayer and its embedded membrane proteins couple protein conformational changes to changes in the packing of the surrounding lipids. The energetic cost of a protein conformational change therefore includes a contribution from the associated bilayer deformation energy (DeltaGdef0), which provides a mechanism for how membrane protein function depends on the bilayer material properties. Theoretical studies based on an elastic liquid-crystal model of the bilayer deformation show that DeltaGdef0 should be quantifiable by a phenomenological linear spring model, in which the bilayer mechanical characteristics are lumped into a single spring constant. The spring constant scales with the protein radius, meaning that one can use suitable reporter proteins for in situ measurements of the spring constant and thereby evaluate quantitatively the DeltaGdef0 associated with protein conformational changes. Gramicidin channels can be used as such reporter proteins because the channels form by the transmembrane assembly of two nonconducting monomers. The monomerleft arrow over right arrow dimer reaction thus constitutes a well characterized conformational transition, and it should be possible to determine the phenomenological spring constant describing the channel-induced bilayer deformation by examining how DeltaGdef0 varies as a function of a mismatch between the hydrophobic channel length and the unperturbed bilayer thickness. We show this is possible by analyzing experimental studies on the relation between bilayer thickness and gramicidin channel duration. The spring constant in nominally hydrocarbon-free bilayers agrees well with estimates based on a continuum analysis of inclusion-induced bilayer deformations using independently measured material constants.
Asunto(s)
Canales Iónicos/química , Membrana Dobles de Lípidos/química , Proteínas de la Membrana/química , Gramicidina/química , Conformación Proteica , Solventes/química , TermodinámicaRESUMEN
In the linear gramicidins, the four aromatic residues at positions 9, 11, 13, and 15 are well-known to be important for the structure and function of membrane-spanning gramicidin channels. To investigate whether the "spacer" residues between the tryptophans in gramicidin A (gA) are important for channel structure and function, D-Leu-10, -12. and -14 of gA were replaced by Ala, Val, or Ile. (For practical reasons, the Ile substitutions were introduced into the enantiomeric gramicidin A-, gA-.) Circular dichroism spectra of [D-Ala10,12,14]gA, [D-Val10,12,14]gA, or [Ile10,12,14]gA- incorporated into sodium dodecyl sulfate micelles or 1, 2-dimyristoyl-sn-glycero-3-phosphocholine vesicles differ from the spectrum of the native [D-Leu10,12,14]gA. All the analogue spectra display reduced ellipticity at both 218 and 235 nm, indicating the presence of double-stranded conformers with the Ala analogue spectra showing the largest departure from the native gA spectra. Size-exclusion chromatograms of the Val and Ile analogues show both monomer and dimer peaks, accompanied by peak broadening; the chromatograms for the Ala analogue show broad, overlapping peaks and suggest the presence of higher oligomers and/or (rapidly) interconverting conformations. All three analogues form membrane-spanning channels, with the channel-forming potency of the Ala analogue being much less than that of gA or the other analogues. In 1.0 M CsCl, the conductance of each analogue channel is approximately 25% less than that of [D-Leu10,12,14]gA channels. The lifetimes of the analogue channels also are less than of [D-Leu10,12, 14]gA channels, with the largest (8-fold) reduction being for [D-Ala10,12,14]gA channels. Hybrid channel experiments show that the beta6.3-helical backbone folding pattern is retained in the channel-forming subunits and that the substitutions primarily influence ion entry. Both the bulk and the stereochemistry of the aliphatic residues between the tryptophans of gA are important for channel structure and function.